The present invention relates to deletion mutants of the IL-6 receptor protein, particularly of the beta chain (gp130) of the IL-6 receptor protein, to DNA encoding said protein as well as to RNA derived therefrom. Moreover, the invention relates to substances which specifically block the binding of gp130 to Hck as well as to pharmaceutical preparations containing said substances in an amount effective to treat multiple myeloma.
IL-6 is an important growth factor for multiple myelomas (MM). For this reason, the mechanisms have been studied by which IL-6 induces cell growth. Previously, we have shown that IL-6 induces the activation and tyrosine phosphorylation of Hck, a tyrosine kinase of the Src family. It has been demonstrated that in MM cell lines Hck binds to the IL-6 receptor xcex2 chain gp130. A series of gp130 deletion mutants has been constructed on the basis of a chimeric receptor comprising the extracellular portion of the erythropoietin receptor and the intracellular portion of gp130. Cloning of gp130 is described in [Hibi M., 1990]. Surprisingly, the deletion of a region of 41 amino acids in length between residues 771 and 811 of gp130 (xcex94771-811) results in complete disruption of Hck binding to gp130. A striking feature of this region is its remarkably high content of negatively charged residues; for this reason it has been also referred to as xe2x80x9cacidic domainxe2x80x9d. Since the xcex94771-811 deletion is localized between the two STAT3 binding sites at residues tyrosine (Y) 814 and 767, point mutations have been generated at these sites; this was done to exclude that Y814 or Y767 affect Hck binding to gp130. STAT3 activation by gp130 was not affected by the xcex94771-811 mutant. Eventually, the stable transfection of receptor mutants into a growth factor-dependent pro B cell line, Baf-B03, showed that deletion xcex94771-811 significantly reduced the proliferative response of cells to gp130 stimulation. In conclusion, it has been shown for the first time that Hck binds to an acidic domain of gp130 which is critical for mediating the proliferative response via growth factor-activated gp130.
Interleukin-6 is a major growth factor for MM cells in vitro and in vivo [Klein B., 1995; Hallek M., 1998; Klein B., 1992; Klein B., 1995; Kawano M., 1988; Zhang X. G., 1989]. Despite evidence for a role of IL-6 in the pathogenesis of MM little is known about the signaling mechanisms responsible for IL-6-mediated cell growth in MM.
To exert these biological effects IL-6 must bind to the IL-6 receptor (IL-6R) composed of two xcex1-chains (IL-6Rxcex1, 80 kDa) and two xcex2 chains, IL-6Rxcex2 or gp130 (130 kDa). Two moieties of IL-6 and two pairs of these receptor chains form a functional hexameric IL-6R complex [Simpson R. J., 1997; Somers W., 1997; Ward L. D., 1996]. The subsequent intracellular signaling events are activated by gp130. Activation of IL-6R stimulates at least two major signal transduction pathways, the Ras/mitogen activated protein kinase (MAPK) signaling cascade [Neumann C., 1996; Ogata A., 1997; Takahashi-Tezuka M., 1998; Shi Z. Q., 1998; Boulton T. G., 1994] and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway [Berger L. C., 1994; Ihle J. N., 1994; Stahl N., 1994; Gerhartz C., 1996]. However, the signaling cascades mediating IL-6 induced cell growth are not well defined. It has been shown earlier that JAK and STAT proteins are activated by IL-6 in MM cells independently of the proliferative response. In contrast, MAPK was only activated in cells and cell lines showing a proliferative response to IL-6 [Ogata A., 1997].
The inventors have shown previously that at least three members of the Src-family of tyrosine kinases, Fyn, Hck, and Lyn, bind to gp130 in MM cells [Hallek M., 1997]. Stimulation of cells with IL-6 increased the activity of these kinases.
It is an object of the present invention to provide means for the inhibition or at least significant reduction of the stimulation of multiple myeloma cells by IL-6 in order to at least significantly decrease the proliferation of tumor cells and particularly of myeloma cells.
According to the present invention there is provided an IL-6 receptor protein having a deletion in the region of the beta chain which at least comprises amino acids 771-811. In further studies, this region may be restricted further to reveal smaller deletion regions within the 771-811 region which inhibit the binding of Hck. Preferably, the binding of Hck to gp130 is reduced by more than 90%. However, it is to be understood that also a larger region of gp130 may be deleted which comprises the amino acid sequence mentioned above or portions thereof. However, such deletion mutants are preferred which comprise the region of amino acids 771-811 or portions thereof.
Claimed according to the present invention is not only the IL-6 receptor protein having the above-mentioned deletion in gp130 but also gp130 itself carrying the deletion in the region of 771-811 or portions thereof. Also deletions exceeding the region of 771-811 which result in an inhibition of the interaction between Hck and gp130 are comprised by the present invention.
Also DNA encoding the IL-6 receptor proteins characterized above or the mutant gp130as well as RNA derived from said DNA is claimed according to the present invention.
According to the invention it has been surprisingly found that deletion of a specific region of gp130 abolishes the binding of Hck kinase. Surprisingly, this deleted region was not localized in the region of homology boxes which are known to be present in several different growth factor receptors and for which the binding of signaling proteins thereto has been shown. Further surprisingly, the deletion mutants of gp130 in the region described in the following exert an unexpectedly marked inhibition of myeloma cell proliferation.
The region of 41 amino acids found by the present inventors may be further restricted by conventional deletion studies. For example, receptor mutants are prepared for this purpose comprising smaller deletions in the region of amino acids 771-811. In the alternative embodiment, peptides are prepared containing smaller regions of the above-mentioned deletion region comprising 41 amino acids; these smaller peptides containing respective partial sequences of the Hck binding domain are used to xe2x80x9cfishxe2x80x9d Hck. In this manner it will be possible to find such amino acids or amino acid regions, respectively, which are required for Hck binding whereby the so-called xe2x80x9cdrug designxe2x80x9d may be accelerated and simplified. Particularly, two regions consisting of 5 and 7 amino acids, respectively, are of interest within the region comprising 41 amino acids, namely the regions STQPL (SEQ ID NO:22) and SEERPED (SEQ ID NO:23).
On the basis of the deletion region found according to the present invention which comprises amino acids 771-811 of the IL-6 receptor protein beta chain the skilled artisan will be able to purposefully select substances blocking the Hck tyrosine kinase binding site on gp130 protein found according to the present invention. Furthermore, substances may be purposefully found which specifically block the gp130 protein binding site on Hck tyrosine kinase. For example, these substances may be proteins, preferably short peptides blocking the docking site, i.e. the protein binding pocket. In other embodiments these may also be inorganic or organic molecules being no proteins in nature.
Starting with the knowledge described in the present application of the binding region of Hck to gp130 it will be possible to develop inhibitors, such as peptides, which fit into the Hck xe2x80x9cbinding pocketxe2x80x9d equally well as Hck or even better and which then block this pocket for Hck, i.e. prevent or at least inhibit the intracellular protein-protein interaction, here each time abbreviated as xe2x80x9cbindingxe2x80x9d, between Hck and gp130. The same object may be achieved by using synthetically prepared substances mimicking Hck or at least the binding region of Hck to gp130 without exerting its function of the promotion of tumor cell growth.
Based on the present invention and using well-known selection techniques the skilled artisan will be able to find such substances which specifically block the binding region of Hck on the beta chain of the IL-6 receptor protein. This means, that the binding site for Hck on gp130 will be masked so that Hck will be unable to recognize and particularly to bind to said binding region.
In an alternative embodiment of the present invention a selection of such substances will be carried out which specifically mask the binding region for gp130 on the Hck tyrosine kinase protein so that Hck will be unable to recognize and particularly to bind to its native binding site on gp130.
In a further embodiment of the present invention peptides will be generated comprising amino acids 771-811 of gp130 or portions of this region which are capable of binding to Hck tyrosine kinase. These peptides may be added to myeloma cells or administered to patients in amounts that competitive binding to the Hck tyrosine kinase occurs whereby the native binding site of Hck on gp130 is competitively blocked so that the number of Hck molecules able to bind to gp130 will be considerably decreased at least to that extent that the proliferation of myeloma cells is inhibited.
The term xe2x80x9cbindingxe2x80x9d according to the present invention means any type of interaction of the Hck tyrosine kinase with gp130which is capable of performing a signal transduction between Hck and gp130. This interaction between Hck and gp130 will be avoided by the deletion mutants described above as well as the inhibitors presented herein.
The substances contemplated for the use in a screening process comprise inorganic and organic molecules wherein the organic molecules are both proteins or peptides and molecules lacking amino acids.
After restricting the binding domain of Hck on gp130 to a small region using the present invention conventional screening processes may be used for a relatively quick and purposeful analysis and selection of molecules in large amounts which bear the features desired according to the present invention. Substances having the desired properties, i.e. which inhibit or at least strongly reduce the specific binding of Hck to gp130, may be processed to form a pharmaceutical preparation which together with conventional pharmaceutical carriers and additives may be used in patients suffering from a tumor, e.g. a multiple myeloma. Using this pharmaceutical preparation it will be possible to significantly inhibit the proliferative growth of tumor cells, e.g. myeloma cells, to achieve a therapeutical effect, i.e. at least an alleviation of the disease.
While it has been known that Hck interacts with gp130, however, it was new to identify the binding domain and above all to find that a deletion of this binding site results in a marked decrease of the cell growth of tumor cells. On the basis of the present invention it is now possible to purposefully develop means for the inhibition or even the abolishment of the interaction between Hck and gp130 using methods known per se.
The knowledge of the binding domain of Hck on the gp130molecule which is highly relevant for the pathogenesis of a tumor disease has been the initial and essential step for a purposeful development of medicaments. Having now found the protein sequence data bases may be screened. For example, natural substances may be found in these data bases which may be used as the blocking agent, or starting from the amino acid sequence the possible three-dimensional structure of the binding pocket may be deduced by means of computer programs. Then, synthetic substances may be prepared directed against the binding pocket which closely fit into said pocket.
Because redundancy is often encountered in nature it may be considered that the Hck binding domain is also present in other molecules with which Hck interacts. Furthermore, the amino acid sequence described in the present invention enables screening for novel and up to now unknown binding partners of Hck. Hck belongs to the family of Src kinases. According to present knowledge this family comprises 9 kinases with high homology to each other all of which are potential or have been identified as proto-oncogenes and are highly likely to participate in the generation of numerous cancer types. On the basis of the binding domain described according to the present invention a search for such binding partners of Hck may be carried out which are new and have not been described up to now.
In the following, the present invention will be described in more detail with respect to Examples and Figures and Tables. The accompanying Figures and Tables show:
Table 1: Schematic overview of gp130 truncation and deletion mutants. Several truncation and deletion mutants of gp130were cloned as chimeric receptor molecules as described in the method section, the numbers indicating amino acids of wild type gp130. The sites of the tyrosine residues and the xe2x80x9cacidic domainsxe2x80x9d are indicated. All of the mutants as well as wild type gp130 (Eg) carry His and myc labels for the expression in Cos-7 cells or His and V5 labels for Baf-B03 cell experiments.
Table 2: Diagram showing internal deletions in the Hck binding region. Several chimeric gp130 mutants with further deletions in the Hck binding region (771-811) were constructed as described. The numbers represent amino acid positions in wild type gp130. The erythropoietiri receptor domain has been underlaid with black, gp130 with light grey, and the STAT3 binding region at tyrosine 814 in dark grey.
Table 3: Point mutations of gp130. Several site specific mutations have been introduced in gp130. The three tyrosine residues in the vicinity of the Hck binding region (771-811) were point mutated to phenylalanine (756, 767, and 814). In addition, a double point mutant (YY759/767FF) was constructed. The DDM mutant (double point and deletion mutant) lacks tyrosines 759 and 767 as well as amino acids 771-827. All of the mutants as well as wild type gp130 (Eg) carry His and myc labels for the expression in Cos-7 cells or His and V5 labels for Baf-B03 cell experiments.